We present a DNA library preparation method that has allowed us to reconstruct a high-coverage (30×) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of “missing evolution” in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans.

https://science.sciencemag.org/content/sci/338/6104/222.full.pdf

A high-coverage genome sequence from an archaic Denisovan individual

How species with similar repertoires of protein-coding genes differ so markedly at the phenotypic level is poorly understood. By comparing organ transcriptomes from vertebrate species spanning ~350 million years of evolution, we observed significant differences in alternative splicing complexity between vertebrate lineages, with the highest complexity in primates. Within 6 million years, the splicing profiles of physiologically equivalent organs diverged such that they are more strongly related to the identity of a species than they are to organ type. Most vertebrate species-specific splicing patterns are cis-directed. However, a subset of pronounced splicing changes are predicted to remodel protein interactions involving trans-acting regulators. These events likely further contributed to the diversification of splicing and other transcriptomic changes that underlie phenotypic differences among vertebrate species.

https://science.sciencemag.org/content/sci/338/6114/1587.full.pdf

The Evolutionary Landscape of Alternative Splicing in Vertebrate Species

Cis-regulatory sequences, such as enhancers and promoters, control development and physiology by regulating gene expression. Mutations that affect the function of these sequences contribute to phenotypic diversity within and between species. With many case studies implicating divergent cis-regulatory activity in phenotypic evolution, researchers have recently begun to elucidate the genetic and molecular mechanisms that are responsible for cis-regulatory divergence. Approaches include detailed functional analysis of individual cis-regulatory elements and comparing mechanisms of gene regulation among species using the latest genomic tools. Despite the limited number of mechanistic studies published to date, this work shows how cis-regulatory activity can diverge and how studies of cis-regulatory divergence can address long-standing questions about the genetic mechanisms of phenotypic evolution.

Cis-regulatory elements: molecular mechanisms and evolutionary processes underlying divergence

Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing.

Ever-Changing Landscapes: Transcriptional Enhancers in Development and Evolution.

Genetic variation within and between species can be shaped by population-level processes and mutation; however, the relative impact of "survival of the fittest" and "arrival of the fittest" on phenotypic evolution remains unclear. Assessing the influence of mutation on evolution requires understanding the relative rates of different types of mutations and their genetic properties, yet little is known about the functional consequences of new mutations. Here, we examine the spectrum of mutations affecting a focal gene in Saccharomyces cerevisiae by characterizing 231 novel haploid genotypes with altered activity of a fluorescent reporter gene. 7% of these genotypes had a nonsynonymous mutation in the coding sequence for the fluorescent protein and were classified as "coding" mutants; 2% had a change in the S. cerevisiae TDH3 promoter sequence controlling expression of the fluorescent protein and were classified as "cis-regulatory" mutants; 10% contained two copies of the reporter gene and were classified as "copy number" mutants; and the remaining 81% showed altered fluorescence without a change in the reporter gene itself and were classified as "trans-acting" mutants. As a group, coding mutants had the strongest effect on reporter gene activity and always decreased it. By contrast, 50%-95% of the mutants in each of the other three classes increased gene activity, with mutants affecting copy number and cis-regulatory sequences having larger median effects on gene activity than trans-acting mutants. When made heterozygous in diploid cells, coding, cis-regulatory, and copy number mutant genotypes all had significant effects on gene activity, whereas 88% of the trans-acting mutants appeared to be recessive. These differences in the frequency, effects, and dominance among functional classes of mutations might help explain why some types of mutations are found to be segregating within or fixed between species more often than others.

/pdf/contrasting-properties-of-gene-specific-regulatory-coding-3so7nks8t8.pdf

Contrasting Properties of Gene-Specific Regulatory, Coding, and Copy Number Mutations in Saccharomyces cerevisiae: Frequency, Effects, and Dominance

cis-regulatory DNA sequences known as enhancers control gene expression in space and time. They are central to metazoan development and are often responsible for changes in gene regulation that contribute to phenotypic evolution. Here, we examine the sequence, function, and genomic location of enhancers controlling tissue- and cell-type specific expression of the yellow gene in six Drosophila species. yellow is required for the production of dark pigment, and its expression has evolved largely in concert with divergent pigment patterns. Using Drosophila melanogaster as a transgenic host, we examined the expression of reporter genes in which either 5′ intergenic or intronic sequences of yellow from each species controlled the expression of Green Fluorescent Protein. Surprisingly, we found that sequences controlling expression in the wing veins, as well as sequences controlling expression in epidermal cells of the abdomen, thorax, and wing, were located in different genomic regions in different species. By contrast, sequences controlling expression in bristle-associated cells were located in the intron of all species. Differences in the precise pattern of spatial expression within the developing epidermis of D. melanogaster transformants usually correlated with adult pigmentation in the species from which the cis-regulatory sequences were derived, which is consistent with cis-regulatory evolution affecting yellow expression playing a central role in Drosophila pigmentation divergence. Sequence comparisons among species favored a model in which sequential nucleotide substitutions were responsible for the observed changes in cis-regulatory architecture. Taken together, these data demonstrate frequent changes in yellow cis-regulatory architecture among Drosophila species. Similar analyses of other genes, combining in vivo functional tests of enhancer activity with in silico comparative genomics, are needed to determine whether the pattern of regulatory evolution we observed for yellow is characteristic of genes with rapidly evolving expression patterns.

/pdf/nomadic-enhancers-tissue-specific-cis-regulatory-elements-of-405kdm2c3o.pdf

Nomadic enhancers: Tissue-specific cis-regulatory elements of yellow have divergent genomic positions among Drosophila species

The regulation of gene expression controls development, and changes in this regulation often contribute to phenotypic evolution. Drosophila pigmentation is a model system for studying evolutionary changes in gene regulation, with differences in expression of pigmentation genes such as yellow that correlate with divergent pigment patterns among species shown to be caused by changes in cis- and trans-regulation. Currently, much more is known about the cis-regulatory component of divergent yellow expression than the trans-regulatory component, in part because very few trans-acting regulators of yellow expression have been identified. This study aims to improve our understanding of the trans-acting control of yellow expression by combining yeast-one-hybrid and RNAi screens for transcription factors binding to yellow cis-regulatory sequences and affecting abdominal pigmentation in adults, respectively. Of the 670 transcription factors included in the yeast-one-hybrid screen, 45 showed evidence of binding to one or more sequence fragments tested from the 5′ intergenic and intronic yellow sequences from D. melanogaster, D. pseudoobscura, and D. willistoni, suggesting that they might be direct regulators of yellow expression. Of the 670 transcription factors included in the yeast-one-hybrid screen, plus another TF previously shown to be genetically upstream of yellow, 125 were also tested using RNAi, and 32 showed altered abdominal pigmentation. Nine transcription factors were identified in both screens, including four nuclear receptors related to ecdysone signaling (Hr78, Hr38, Hr46, and Eip78C). This finding suggests that yellow expression might be directly controlled by nuclear receptors influenced by ecdysone during early pupal development when adult pigmentation is forming.

Potential Direct Regulators of the Drosophila yellow Gene Identified by Yeast One-Hybrid and RNAi Screens

cis -regulatory sequences known as enhancers play a key role in regulating gene expression. Evolutionary changes in these DNA sequences contribute to phenotypic evolution. The Drosophila yellow gene, which is required for pigmentation, has emerged as a model system for understanding how cis -regulatory sequences evolve, providing some of the most detailed insights available into how activities of orthologous enhancers have diverged between species. Here, we examine the evolution of yellow cis -regulatory sequences on a broader scale by comparing the distribution and function of yellow enhancer activities throughout the 59 intergenic and intronic sequences of Drosophila melanogaster , Drosophila pseudoobscura , and Drosophila willistoni. We find that cis -regulatory sequences driving expression in a particular tissue are not as modular as previously described, but rather have many redundant and cryptic enhancer activities distributed throughout the regions surveyed. Interestingly, cryptic enhancer activities of sequences from one species often drove patterns of expression observed in other species, suggesting that the frequent evolutionary changes in yellow expression observed among Drosophila species may be facilitated by gaining and losing repression of pre-existing cis -regulatory sequences.

https://www.genetics.org/content/genetics/early/2019/03/06/genetics.119.301985.full.pdf

Gizem Kalay

Papers

Cis-regulatory elements: molecular mechanisms and evolutionary processes underlying divergence

Contrasting Properties of Gene-Specific Regulatory, Coding, and Copy Number Mutations in Saccharomyces cerevisiae: Frequency, Effects, and Dominance

Nomadic enhancers: Tissue-specific cis-regulatory elements of yellow have divergent genomic positions among Drosophila species

Potential Direct Regulators of the Drosophila yellow Gene Identified by Yeast One-Hybrid and RNAi Screens

Redundant and Cryptic Enhancer Activities of the Drosophila yellow Gene.